The conversion of fluid blood to a blood clot, a gelatinous mass which causes the sealing of injured blood vessels by clot formation, occurs in blood clotting. Thereby, the conversion of the soluble fibrinogen present in plasma to the fibrous, gelatinous coagulation material, fibrin, occurs in a multi-step process (the so-called blood coagulation cascade) in which at least 15 different blood coagulation factors, which are characterized with roman numerals, are involved, each of which, when activated, activates the next respective inactive step.
Among the blood factors, calcium ions (Factor IV), fibrinogen (Factor I) and prothrombin (Factor II) continuously circulate in the blood, others are activated by tissue injury (Factor III) or contact with collagen or phospholipids from thrombocytes (Factor XII). Several serine proteases, such as kallikrein, thrombin and the activated Factors VII, IX, X and XI, are found among the remaining blood clotting factors.
In the presence of von Willebrand Factor (a component of clotting Factor VIII), thrombocytes cling to the collagen of injured connective tissue by adhesion. They change their form and develop protrusions, and in addition to this, their outer membrane facilitates the adhesion of further thrombocytes. Thereafter, various substances are released from their granula, whereby vessel constriction as well as accumulation and activation of other factors of plasmic blood clotting are brought about.
In hemophilia (bleeder's disease), blood clotting is disturbed by a lack of certain plasmic blood clotting factors. In hemophilia A, the tendency to bleed is caused by a lack of Factor VIII; in hemophilia B, a lack of Factor IX. Thereby, either the synthesis of the Factor protein can be decreased or a defective molecule with reduced activity is formed. The treatment of hemophilia occurs by replacement of the missing clotting factor by factor concentrates from blood conserves.
Several of the proteins involved in human blood clotting possess an affinity for metal ions, such as Ca.sup.2+ ions. This affinity is absolutely essential for the function of the clotting factors. The binding occurs through glutamic acid residues; thereby, several glutamic acid residues (Glu) of the N-terminal Gla region of various clotting factors are converted to 4-carboxy-L-glutamic acid (Gla) in a vitamin K dependent reaction (see A. Tulinsky, Thromb. Haemost. 66 (1991) 16-31). These Gla residues then bring about the binding of divalent metal ions (see B. Furie and B. C. Furie, Cell 53 (1988) 508-518).
In the biosynthesis of vitamin K dependent clotting factors in humans, a precursor molecule is first formed whose N-terminus has an additional pre-pro-sequence.
The pre-pro-sequence represents a signal sequence which causes the oriented transport of the protein in the cell. This pre-sequence is cleaved in secretion of the protein from the cell. The pro-sequence consists of about 15 to 18 amino acids and serves as a recognition sequence in the conversion of the glutamic acid residues to 4-carboxy-L-glutamic acid. After successful carboxylation, the pro-sequence is also cleaved. If the pro-sequence is not cleaved or only incompletely cleaved, only low activity clotting factors result. Human Factor IX has a molecular weight of about 55,000 Dalton. Its pro-sequence consists of 18 amino acids, whereby the molecular weight is increased by about 2000 Dalton. In the purification of Factor IX from plasma, active Factor IX is almost exclusively obtained. The purification of Factor IX from plasma is, however, very difficult because Factor IX is only present in low concentration in plasma (5 .mu.g/ml; see L. O. Andersson, Thrombosis Research 7 (1975) 451-459).
Therefore, it is desirable to have recombinant Factor IX made available for the treatment of patients affected with hemophilia.
The DNA sequence of Factor IX used for expression also comprises the pre-pro-sequences. It is expected from the expressing cell systems that they quantitatively cleave these sequences for complete processing of Factor IX and secrete a physiologically active clotting factor. However, in the case of Factor IX, it has been determined that the inherent potential of transformed cells for cleaving the pro-sequence is not sufficient. Therefore, various efforts for the production of recombinant Factor IX have led to products with only low activity (R. J. Kaufman et al., J. Biol. Chem. 261 (1986) 9622-9628; S. Busby et al., Nature 316 (1985) 217-273; D. J. G. Rees et al., EMBO J. 7 (1988) 2053-2061). This can be traced back to an incomplete cleavage of the pro-sequence (P. Meulien et al., Prot. Engineer. 3 (1990) 629-633) because a mixture of recombinantly produced pro-Factor IX and Factor IX is present in cell supernatants.
Up to now, an improvement in the recovery of recombinant, physiologically active Factor IX could only be achieved through genetic manipulation of the pro-sequence. It has thus been attempted to couple the pro-sequence of Factor VII to the DNA sequence of Factor IX in order to obtain a more effective cleavage of the pro-sequence (K. Berkner et al., Current Advances in Vitamin K Research, Elsevier Science Publishing Co., Inc. (1988) 199-207). P. Meulien et al., Prot. Engineer. 3 (1990) 629-633) examined the influence of mutations in the region of the pro-peptide cleavage site of Factor IX. They determined that the yield of active Factor IX can be distinctly increased by introduction of a point mutation in position +1 (alanine versus tyrosine); in comparison with wild-type Factor IX, which demonstrates a specific activity of 45-55% after purification over a DEAE-Sepherodex.RTM. column and stepwise elution with 0.3 M NaCl in the physiological pH range, a specific activity of 85 to 100% was found for the mutated Factor IX.